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Live-Imaging Technique Goes Sharper, Deeper, Faster

Photonics.comJul 2011
PASADENA, Calif., July 29, 2011 — Researchers from the California Institute of Technology have developed a novel approach that could redefine optical imaging of live biological samples by simultaneously achieving high resolution, high penetration depth and high imaging speed.

For modern biologists, the ability to capture high-quality 3-D images of living tissues or organisms over time is necessary to answer problems in areas ranging from genomics to neurobiology and developmental biology. The better the image, the more detailed the information that can be drawn from it.

Three-dimensional live imaging of zebra fish (upper panel) and fruit fly (lower panel) embryos with two-photon light-sheet microscopy. The upper panel shows not only the embryo's tissue (in white) but also green fluorescent protein (GFP) driven by a gene-specific promoter (in orange). The lower panel shows the individual cell nuclei of the fruit fly embryo, labeled with GFP, imaged at different time points during its embryonic development. [Image: Willy Supatto, Seth Ruffins and Thai Truong, Caltech]
"Before our work, the state-of-the-art imaging techniques typically excelled in only one of three key parameters: resolution, depth or speed. With our technique, it's possible to do well in all three and, critically, without killing, damaging or adversely affecting the live biological samples," said biologist Scott Fraser, director of the Biological Imaging Center at Caltech's Beckman Institute. He is the senior author of the paper describing the technique, which appears in the July 14 online edition of the journal Nature Methods.

The research team achieved this imaging hat trick by first employing an unconventional method called light-sheet microscopy, where a thin, flat sheet of light illuminates a biological sample from the side, creating a single lit optical section through the sample. The light given off by the sample is then captured with a camera oriented perpendicularly to the light sheet, harvesting data from the entire illuminated plane at once. This allows millions of image pixels to be captured simultaneously, reducing the light intensity required for each pixel. This not only enables fast imaging speed but also decreases the amount of light-induced damage to the living samples, which the teams demonstrated using the embryos of fruit flies and zebra fish.

To achieve sharper image resolution with light-sheet microscopy deep inside the biological samples, the team used two-photon excitation for the illumination. This process has been used previously to allow deeper imaging of biological samples; however, it usually is used to collect the image one pixel at a time by focusing the exciting light to a single small spot.

"The conceptual leap for us was to realize that two-photon excitation could also be carried out in sheet-illumination mode," said Thai Truong, a postdoctoral fellow in Fraser's laboratory. This novel side-illumination with two-photon excitation is the subject of a pending patent.

"We did not want to develop a fanciful optical imaging technique that excels only in one niche area, or that places constraints on the sample so severe that the applications will be limited,” Truong said. “With a balanced high performance in resolution, depth and speed, all achieved without photodamage, two-photon light-sheet microscopy should be applicable to a wide variety of in vivo imaging applications."

"We believe the performance of this imaging technique will open up many applications in life sciences and biomedical research — wherever it is useful to observe, noninvasively, dynamic biological process in 3-D and with cellular or subcellular resolution," said Willy Supatto, another co-author of the paper and a former postdoctoral fellow in Fraser's laboratory (now at the Centre National de la Recherche Scientifique in France).

1. In optics, the ability of a lens system to reproduce the points, lines and surfaces in an object as separate entities in the image. 2. The minimum adjustment increment effectively achievable by a positioning mechanism. 3. In image processing, the accuracy with which brightness, spatial parameters and frame rate are divided into discrete levels.